Glucose is an ancient and central signaling molecule in a broad range of organisms from E. coli, yeast to plants and humans. Despite the essential roles of glucose in gene expression, physiology, metabolism, cell proliferation and death, growth and development, and human diseases, the molecular and cellular mechanisms of glucose signaling remain mostly elusive in plants and animals. Our research in the model plant Arabidopsis thaliana has provided compelling genetic and biochemical evidence that hexokinase1 (HXK1) is an evolutionarily conserved glucose sensor that integrates nutrient and hormone signals to govern gene expression and plant growth in response to environmental cues. How the metabolic enzyme mediates glucose signaling remains a mystery. Our recent findings indicate that Arabidopsis HXK1 mediates glucose signaling without its metabolic activity, and interacts with two HXK1 Unconventional Partners (HUP1: a scaffolding protein interacting with HXK1, HUP2 and transcription factors (TFs);HUP2: an ATPase interacting with HXK1, HUP1 and other ATPases) in the nucleus to control transcription and diverse glucose responses. The goal of this research project is to elucidate the mechanisms of glucose signaling controlled by the nuclear sensor HXK1 in Arabidopsis. The proposed experiments aim to use molecular, cellular, genetic, and genomic approaches to elucidate HXK1 functions and regulation, and to analyze two sets of signaling regulators acting downstream of the nuclear HXK1-HUP1/2 complex. The project will focus on characterizing and integrating functions of eight Arabidopsis genes in glucose signaling using transgenic plants and loss-of-function mutants with specific defects in the glucose sensor HXK1, HXK1 signaling partners HUP1, novel transcription factors (MYB, SCL3, ZFP) and ATPases (HUP2/RPT5, RPT2 and RPT6). Near whole-genome microarray and chromatin-immunoprecipitation (ChIP) will be carried out to identify the primary HXK1 target genes and characterize their regulation in vivo. The project challenges the existing paradigm on metabolism-based glucose responses in plants and animals, and aims to build a new conceptual framework to enhance our understanding of the molecular and cellular mechanisms of glucose signaling from plants to humans. Three Specific Aims are: Aim 1. Elucidate the functions and regulation of the nuclear glucose sensor HXK1 Aim 2. Characterize three novel TFs in HXK1-HUP1/2-mediated glucose signaling Aim 3. Define functions of three ATPases in the nuclear RPT5/2/6 complex. PUBLIC HEALTH RELEVANCE: In spite the central roles of glucose in gene expression, physiology, metabolism, cell proliferation and death, growth and development, and human diseases, the molecular mechanisms of glucose signaling remain elusive in plants and animals. Our recent research has provided compelling evidence that glucose signaling can be uncoupled from glucose metabolism in multicellular eukaryotes, and Arabidopsis hexokinase1 acts as an evolutionarily conserved sensor playing a pivotal role in nuclear glucose signaling. The proposed research will discover novel regulators and elucidate their functions in nuclear glucose signaling, and provide innovative tools for future improvement of agriculture, environment and renewable energy production, as well as human health in treating diabetes, obesity, cancer and ageing modulated by glucose signaling.